Cochleates made with soy phosphatidylserine

ABSTRACT

Unpurified or low pure soy phosphatidylserine is used to make cochleates. The cochleates contain about 40-74% soy phosphatidylserine, a multivalent cation and a biological active. A preferred cochleate contains the antifungal agent amphotericin B.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/677,414 filed on Jul. 30, 2012 and U.S. Provisional ApplicationNo. 61/835,825 filed Jun. 17, 2013, the entire contents of eachaforementioned application are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the ability of unpurified or low purity(40-74% by weight) soy-based phosphatidylserine (PS) to preparecochleates, methods of preparing drug-cochleates from soy-based PS andthe use of this drug-loaded cochleate as a pharmaceutical treatment.

BACKGROUND OF THE INVENTION

Cochleate delivery vehicles are a broad-based technology for thedelivery of a wide range of bioactive therapeutic products. Cochleatedelivery vehicles are stable phospholipid-cation precipitates composedof simple, naturally occurring materials, for example,phosphatidylserine and calcium.

The bilayer structure of cochleates provides protection from degradationfor associated, or “encochleated,” molecules. Since the entire cochleatestructure is a series of solid layers, components within the interior ofthe cochleate structure remain substantially intact, even though theouter layers of the cochleate may be exposed to harsh environmentalconditions or enzymes. This includes protection from digestion in thestomach.

Taking advantage of these unique properties, cochleates have been usedto mediate and enhance the oral bioavailability of a broad spectrum ofimportant but difficult to formulate biopharmaceuticals, includingcompounds with poor water solubility, protein and peptide drugs, andlarge hydrophilic molecules. For example cochleate-mediated oraldelivery of amphotericin B, large DNA constructs/plasmids for DNAvaccines and gene therapy, peptide formulations, and antibiotics such asclofazimine has been achieved.

Cochleates can be stored in cation-containing buffer, or lyophilized toa powder, stored at room temperature, and reconstituted with liquidprior to administration. Lyophilization has no adverse effects oncochleate morphology or functions. Cochleate preparations have beenshown to be stable for more than two years at 4° C. in acation-containing buffer, and at least one year as a lyophilized powderat room temperature.

Cochleates can be prepared by several methods, such as trapping orhydrogel methods (International Application Publication No. WO03/082209, the entire content of which is incorporated herein byreference).

Soy PS is sold in health food stores as a nutritional supplement.Non-purified (40%) PS has been used and studied as a nutritionalsupplement and as a component that has a beneficial effect on enhancingthe brain functions in elderly people (Villardita C et al., Clin. TrialsJ. 24, 1987, 84-93).

Although non-purified soy PS (NSPS) has been sold and studied onpatients, NSPS (or low purity PS) has never been used to make cochleatesand to deliver a drug using these cochleates. As previously disclosed inWO 03/082209, NSPS does not form cochleates and that a purificationprocess is needed to enhance the NSPS in the content of PS, until atleast about 75% by weight of PS is reached, such percentage allowing theformation of cochleates.

SUMMARY OF THE INVENTION

It has been unexpectedly found that NSPS or low purity soy-based PS(40-74% by weight) can still form cochleates.

Briefly, in accordance with the present invention, improved lipid basedcochleates are made by using non-purified or low purity soyphosphatidylserine as the lipid source. The improved cochleates containsoy phosphatidylserine in an amount of about 40%-74% (preferably 45-70%,more preferably 45-55%) by weight of the lipid. The improved cochleatescan be empty or loaded cochleates. Loaded cochleates can contain anybiological actives or combination of biological actives such as, forexample, a protein, a small peptide, a polynucleotide, anaminoglycoside, an antiviral agent, an anesthetic, an antibiotic, anantifungal, an anticancer, an immunosuppressant, a steroidalanti-inflammatory, a non-steroidal anti-inflammatory, a tranquilizer, anutritional supplement, an herbal product, a vitamin or a vasodilatoryagent. Of particular interest in practicing the present invention,antifungal agents or antibiotic agents are loaded into the presentsoy-based phosphatidylserine cochleates to provide a cost effective andimproved antifungal drug/antibiotic drug with reduced toxicity.Preferred antifungal agents include amphotericin-B and nystatin.Preferred antibiotic agents include an aminoglycoside and amikacin.

The improved lipid based cochleates of the present invention can be madeby a method comprising the steps of: (a) preparing liposomes in anaqueous medium wherein the liposomes have (i) a lipid bilayer comprisingsoy-based phosphatidylserine in an amount of about 40%-74% (preferably45-70%, more preferably 45-55%) by weight of the lipid bilayer and (ii)a load of a biological active; (b) adding a multivalent cation to thesuspension of liposomes of (a) to form the soyphosphatidylserine/biological active cochleates; and (c) collecting thesoy-based phosphatidylserine/biological active cochleates.

The present invention also teaches that the soyphosphatidylserine/biological active cochleates can be administered topatients with fungal infections or with bacterial infections. Thepresent soy phosphatidylserine/biological active cochleates areconveniently administered orally even in the treatment of systemicfungal infections of immune compromised patients. The presentphosphatidylserine/biological active cochleates are also administeredparenterally, or by other means of administration. The preferredbiological active is amphotericin-B, curcumin, and amikacin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows amikacin cochleate in vitro study against MAC 101 and MAC109 in mouse peritoneal macrophage.

FIG. 2 shows amikacin cochleate in vivo efficacy in a mouse model of MAC101 infection IP-delivery.

FIG. 3 shows amikacin cochleate in vivo efficacy in a mouse model of MAC101 infection oral-delivery.

FIG. 4 shows gentamicin encochleation related to PS properties.

FIG. 5 shows encochleation efficiency of amikacin bile salts related toPS properties.

FIG. 6 shows encochleation efficiency of amikacin bile salts related toPS properties.

FIG. 7 shows encochleation efficiency of gentamycin bile salts relatedto PS properties.

FIG. 8 shows encochleation efficiency of gentamycin bile salts relatedto PS properties.

FIG. 9 shows encochleation efficiency of paromomycin bile salts relatedto PS properties.

FIG. 10 shows encochleation efficiency of paromomycin bile salts relatedto PS properties.

FIG. 11 shows encochleation efficiency of paromomycin bile salts relatedto PS properties.

DETAILED DESCRIPTION OF THE INVENTION

The following terms when used herein will have the definitions givenbelow.

A “cochleate” is a stable, phospholipid-cation precipitate that can beeither empty or loaded.

An “empty cochleate” is a cochleate that is comprised only ofphospholipid and cations.

A “loaded cochleate” is a cochleate that has one or more biologicalactive compounds within the phospholipid-cation structure.

“Soy phosphatidylserine” or “soy-based phosphatidylserine” isphosphatidylserine that has been derived from a soy based composition.

In practicing the present invention improved phospholipid basedcochleates are made by using soy phosphatidylserine in an amount of40%-74% by weight of the lipid component of the cochleates.Alternatively, the soy phosphatidylserine can be about 40%, 45%, 50%,55%, 60%, 65%, or 70%, or any incremental value thereof, by weight ofthe lipid component of the cochleates. It is to be understood that allvalues, and ranges between these values and ranges are meant to beencompassed by the present invention. In a preferred embodiment thephospholipid comprises 45-70% soy phosphatidylserine. In a morepreferred embodiment, the phospholipid comprises 45-55% soyphosphatidylserine.

Phosphatidic acid is a preferred phospholipid when there is anadditional phospholipid besides phosphatidylserine in the presentlyimproved cochleates. Other phospholipids in addition to phosphatidicacid that can be used in the presently improved cochleates includephosphatidylcholine, phosphatidylinositol and phosphatidylglycerol.Mixtures of the additional phospholipids can also be used in combinationwith the soy phosphatidylserine.

The soy phosphatidylserine starting material is commercially available,or can be purified from soy phospholipid composition, which are mixturesof several soy phospholipids, according to well known and standardpurification techniques.

Any multivalent compound can be used to precipitate the cochleates fromthe liposome starting materials. Preferably, the multivalent compoundsare divalent cations such as Ca⁺⁺, Zn⁺⁺, Ba⁺⁺, and Mg⁺⁺. Preferredsources of these cations include the chloride salts of calcium, zinc,barium, and magnesium. CaCl₂ is a particularly preferred source ofdivalent cations.

In one embodiment, the present soy phosphatidylserine cochleates mayfurther comprise bile salts. The weight ratio of soy-based phospholipidto the bile salts is between 20:1 and 0.5:1, preferably, between 10:1and 3:1.

Bile salts are bile acids compounded with a cation, usually sodium. Bileacids are steroid acids found predominantly in the bile of mammals. Bilesalts are commercially available (for example, Sigma Aldrich catalog#48305 Fluka cholic acid sodium salt, 50% deoxycholic acid sodium salt,50%).

It has been unexpectedly found that the addition of bile salts enhancesthe encochleation efficiency of the soy phosphatidylserine cochleates.For example, with inclusion of bile salts, the soy phosphatidylserinecochleates of the present invention (e.g., with the use of 50% soy PS)are more efficient at encochleating than cochleates containing at leastabout 75% soy phosphatidylserine.

In one embodiment the present soy phosphatidylserine cochleates are madeby a process which comprises the steps of:

a. preparing liposomes in an aqueous medium wherein the liposomes have(i) a lipid bilayer comprising soy-based phosphatidylserine in an amountof about 40%-74% (preferably 45-70%, more preferably 45-55%) by weightof the lipid bilayer and (ii) a load of a biological active;

b. adding a multivalent cation to the suspension of liposomes of (a) toform the soy phosphatidylserine/biological active cochleates; and

c. collecting the soy-based phosphatidylserine/biological activecochleates.

In one embodiment, the aqueous medium containing the suspension ofliposome is a buffered environment having a pH of 6.5-7.5, and the loadof the biological active is at pH 10 or higher prior to addition to theliposomes. In a preferred embodiment, the suspension of liposomes isbuffered with phosphate.

In another embodiment, the method further comprises a step of addingbile salts to the suspension of liposomes of (a) before step (b) oradding bile salts to the soy-based phosphatidylserine/biological activecochleates after step (b), wherein the weight ratio of the lipid bilayerto the bile salts is between 20:1 and 0.5:1, preferably, between 10:1and 3:1. The present invention provides a geodate composition whichcontains (1) a lipid monolayer including a soy-based phospholipid thatcomprises about 40%-74% (preferably 45-70%, more preferably 45-55%) byweight soy phosphatidylserine, disposed about a hydrophobic domain; and(2) a lipid strata disposed about the lipid monolayer, wherein the lipidstrata comprises a structure of alternating cationic layers comprising adivalent cation and negatively charged lipid sheet-like layers; and acargo moiety associated with the hydrophobic domain.

International Patent Application Publication No. WO 2004/041247 hasdisclosed a process to make a geodate composition, the entire content ofwhich is incorporated herein by reference.

The bioactive active/drug (referred to as “load” or drug) can behydrophobic in aqueous media, hydrophilic or amphiphilic. The drug canbe, but is not limited to, a protein, a small peptide, a bioactivepolynucleotide, an antifungal agent, an antiviral agent, an anesthetic,an anti-infectious agent, an antifungal agent, an anticancer agent, animmunosuppressant, a steroidal anti-inflammatory, a nutritionalsupplement, an herbal product, a vitamin, a non-steroidalanti-inflammatory, a tranquilizer or a vasodilatory agent. Examplesinclude Amphotericin B, acyclovir, adriamycin, vitamin A, cabamazepine,melphalan, nifedipine, indomethacin, naproxen, estrogens, testosterones,steroids, phenytoin, ergotamines, cannabinoids rapamycin, propanidid,propofol, alphadione, echinomycine, miconazole nitrate, teniposide,taxanes, paclitaxel, and taxotere.

The drug can be a polypeptide such as cyclosporin, angiotensin I, II andIII, enkephalins and their analogs, ACTH, anti-inflammatory peptides I,II, III, bradykinin, calcitonin, b-endorphin, dinorphin, leucokinin,leutinizing hormone releasing hormone (LHRH), insulin, neurokinins,somatostatin, substance P, thyroid releasing hormone (TRH) andvasopressin.

The drug can be an antigen, but is not limited to a protein antigen. Theantigen can also be a carbohydrate or DNA. Examples of antigenicproteins include envelope glycoproteins from influenza or Sendaiviruses, animal cell membrane proteins, plant cell membrane proteins,bacterial membrane proteins and parasitic membrane proteins.

The antigen is extracted from the source particle, cell, tissue, ororganism by known methods. Biological activity of the antigen need notbe maintained. However, in some instances (e.g., where a protein hasmembrane fusion or ligand binding activity or a complex conformationwhich is recognized by the immune system), it is desirable to maintainthe biological activity. In these instances, an extraction buffercontaining a detergent which does not destroy the biological activity ofthe membrane protein is used. Suitable detergents include ionicdetergents such as cholate salts, deoxycholate salts and the like orheterogeneous polyoxyethylene detergents such as Tween, BRIG or Triton.

Utilization of this method allows reconstitution of antigens, morespecifically proteins, into the liposomes with retention of biologicalactivities, and eventually efficient association with the cochleates.This avoids organic solvents, sonication, or extreme pH, temperature, orpressure all of which may have an adverse effect upon efficientreconstitution of the antigen in a biologically active form.

The presently improved cochleates can include loads with multipleantigenic molecules, biologically relevant molecules or drug formulariesas appropriate.

To isolate the cochleate structures and to remove the polymer solution,cochleate precipitates are repeatedly washed with a buffer containing apositively charged molecule, and more preferably, a divalent cation.Addition of a positively charged molecule to the wash buffer ensuresthat the cochleate structures are maintained throughout the wash step,and that they remain as precipitates.

The medium in which the cochleates are suspended can contain salt suchas sodium chloride, sodium sulfate, potassium sulfate, ammonium sulfate,magnesium sulfate, sodium carbonate. The medium can contain polymerssuch as Tween 80 or BRIG or Triton. The drug-cochleate is made bydiluting into an appropriate pharmaceutically acceptable carrier (e.g.,a divalent cation-containing buffer).

The cochleate particles can be enteric. The cochleate particles can beplaced within gelatin capsules and the capsule can be enteric coated.

The skilled artisan can determine the most efficacious and therapeuticmeans for effecting treatment practicing the instant invention.Reference can also be made to any of numerous authorities and referencesincluding, for example, “Goodman & Gillman's, The Pharmaceutical Basisfor Therapeutics”, (6^(th) Ed., Goodman et al., eds., MacMillan Publ.Co., New York, 1980).

The improved soy phosphatidylserine cochleates of the present inventioncontaining a biological active are conveniently administered to patientsorally whereby the cochleates are absorbed into the bloodstream and thebioactive loads are delivered systemically. This is a particularadvantage for water insoluble drugs such as amphotericin-B andpaclitaxel. Additionally, the toxicity of many hydrophobic drugs issubstantially reduced as seen with soy phosphatidylserine cochleatescontaining amphotericin-B as the load.

The following examples illustrate the practice of the present inventionbut should not be construed as limiting its scope.

Example 1 Amphotericin B Crystal Cochleate Formulation Procedures ofAmphotericin B Crystal Formulation in Bench Size: #1.1 ExperimentFormulation:

43 mg (actually 40 mg Amphotericin B based on the potency assay with theconcentration of 0.932 mg/mg) of Amphotericin B in 1.33 mL 0.1N NaOH wasmixed with 200 mg dioleoyl phosphatidylserine (DOPS, from Avanti or NOFCorporation) in 6.6 mL 50 mM phosphate buffer pH 7.4 (liposome wasfiltered through 5 μm filter) to form liposomes containing theAmphotericin B. 29.3 mg calcium chloride was then added into theresultant mixture to form crystal cochleates. To make nice crystallinecochleates, the lipid:Amphotericin B weight ratio was set to about 5:1.

#1.2 Experiment Formulation:

18 mg Amphotericin B in 0.6 mL 0.1N NaOH was then mixed with 90 mg DOPS(from Avanti or NOF Corporation) in 3.0 mL 50 mM phosphate buffer pH 7.4(liposome was filtered through 5 μm, 8 μm, and 4.5 μm filter) to formliposomes containing the Amphotericin B. 0.33 mL 0.5M calcium chloridesolution was then added into the resultant mixture to form crystalsAmphotericin B cochleates. To make nice crystalline cochleates, thelipid:Amphotericin B weight ratio was about 5:1.

Procedures of Amphotericin B (Amphotericin B) Crystals Cochleates inSmall Scale Size: #1.3 Experiment Formulation:

399 mg (actually 372 mg based on the potency assay with theconcentration of 0.932 mg/mg) Amphotericin B in 12.4 mL 0.1N NaOHsolution was combined with 1.86 g of 50% soy PS (American LecithinCompany or Lipoid LLC) in 62 mL 50 mM phosphate buffer pH 7.4 (liposomewas filtered through 5 μm filter) to form liposomes containing theAmphotericin B. In order to add an antioxidant the suspension, 372 mgvitamin E in 3.72 mL ethyl alcohol was then added into the mixture ofthe liposomes. 520 mg powder of calcium chloride was then added into theresultant mixture to form Amphotericin B crystals cochleates. To makenice crystalline cochleates of Amphotericin B, the lipid:Amphotericin Bweight ratio was about 5:1.

Procedures of Amphotericin B Crystal Formulation Scale up to 4.5 L: #1.4Experiment Formulation:

22.75 g Amphotericin B (actually 21.2 g Amphotericin B based on thepotency assay with the concentration of 0.932 mg/mg) in 707 mL 0.1N NaOHsolution was then mixed with 106 g of 50% soy PS in 3.533 L 50 mMphosphate buffer pH 7.4 (liposome was filtered through 10 μm filter) toform liposomes containing the Amphotericin B. To make a stablesuspension of cochleates, 372 mg vitamin E in 3.72 mL ethyl alcohol wasthen added into the mixture. 194.8 mL 1M calcium chloride was then addedinto the resultant mixture to form crystal cochleates. The final productof cochleates was lyophilized with Freezer Dryer for a few days. To makenice crystalline cochleates of Amphotericin B, the lipid:drug weightratio was set to about 5:1.

Example 2 Curcumin (Granular) Geode Cochleates Formulation Procedures ofCurcumin (Granular) Geode Cochleates in Small Scale Size: #2.1Experiment Formulation:

50 mg curcumin in 1 g castor oil and 4.0 mL ethyl alcohol was dissolvedfirst then combined with 19 of 75% soy PS (American Lecithin Company orLipoid LLC) in 100 mL sterile water (liposome was filtered through Sumfilter) to form liposomes containing the curcumin. In order to decreasethe stickiness of suspension and batter diffusion, 500 mg bovine serumalbumin (“BSA”, or casein) was then added into the mixture of theliposome. 16.9 mL 0.1M calcium chloride was then added into theresultant mixture to form curcumin geode cochleates. The final productof goede cochleates was lyophilized with a freezer dryer for a few days.To make nice geode cochleates of curcumin, the lipid:drug weight ratiowas set to about 20:1.

#2.2 Experiment Formulation:

1.00 mg 75% soy PS in 100 mg sterile water (liposome was filteredthrough Sum filter) was mixed with 500 mg casein (or BSA) and then mixedwith 50 mg, curcumin in 2.0 g castor oil and 4.0 mL ethyl alcohol toform liposome containing the curcumin: 16.9 mL 0.1M calcium chloride wasthen added into the resultant mixture to form geode cochleates. Thefinal product of goede cochleates was lyophilized with a freezer dryerfor a few days. To make nice geode cochleates of curcumin, thelipid:drug weight ratio was set to about 20:1.

Procedures of Curcumin (Granular) Crystal Formulation in Small ScaleSize: #2.3 Experiment Formulation:

1000 mg 75% soy PS in 100 mL sterile water was (liposome was filteredthrough Sum filter) combined with 20 mg curcumin in 4.0 mL ethyl alcoholto form liposome containing the curcumin. In order to help the drugstable in the lipid bilayer, 500 mg casein (or BSA) was then added intothe suspension of the liposome. 16.9 mL 0.1M calcium chloride was thenadded into the resultant mixture to form crystal cochleates. The finalproduct of crystal cochleates was lyophilized with Freezer Dryer for afew days. To make nice crystalline cochleates, the lipid:curcumin weightratio was set to about 50:1.

#2.4 Experiment Formulation:

1000 mg 75% soy PS in 100 mL sterile water was (liposome was filteredthrough 5 μm filter) combined with 20 mg curcumin in 4.0 mL ethylalcohol to form liposome containing the curcumin. 16.9 mL 0.1M calciumchloride was then added into the resultant mixture to form crystalcochleates. In order to help the drug stable in the lipid bilayer, 500mg casein (or BSA) was then added into the suspension of the cochleates.The final product of crystal cochleates was lyophilized with a FreezerDryer for a few days. To make nice crystalline cochleates, thelipid:curcumin weight ratio was set to about 50:1.

Example 3 Amphotericin B (Amphotericin B) Geode Formulation Proceduresof Amphotericin B (Amphotericin B) Geode Formulation in Bench Size: #3.1Experiment Formulation:

7.5 mg Amphotericin B (actually 7 mg based on the potency assay with theconcentration of 0.932 mg/mg) In 0.2 mL 0.1N NaOH solution was mixedwith 50 mg of castor oil and then combined with 35 mg of 50% soy PS in1.75 mL sterile water (liposome was filtered through 5 μm filter) toform geode liposomes containing the Amphotericin B. To prevent thestickiness of Amphotericin B geode formulation, 21 mg BSA or casein wasthen added into the mixture of the Amphotericin B geode liposome. Toincrease the stability of the Amphotericin B geode formulation, 7 mgvitamin E in 70 μL Ethyl alcohol was then added into the mixture of thegeode liposome. To the resultant mixture, 127 μL of 0.5M calciumchloride was then added to form Amphotericin B geode cochleates. To makea nice geode cochleates of Amphotericin B, the lipid:Amphotericin Bweight ratio was about 5:1.

Procedures of Amphotericin B Geode Formulation Scale Up to 150 mL: #3.2Experiment Formulation:

536 mg Amphotericin B (actually 500 mg based on the potency assay withthe concentration of 0.932 mg/mg) In 14.3 mL 0.1N NaOH solution was thenmixed with 3.57 g castor oil and then combined with 2.5 g of 50% soy PSin 125 mL sterile water (liposome was filtered through 5 μm filter) toform geode liposomes containing the Amphotericin B. To prevent thestickiness of the geode formulation, 1.5 g BSA or casein was then addedinto the mixture of geode liposome. To increase the stability of theAmphotericin B geode formulation, 250 mg of vitamin E in 2.5 mL ethylalcohol was then added into the mixture of the geode liposome. To theresultant mixture was then added 8.88 mL of 0.5M calcium chloride toform geode cochleates. The pH of the final mixture was adjusted toneutral with 0.3 mL 1N HCl. To make nice geode cochleates ofAmphotericin B, the lipid:Amphotericin B weight ratio was about 5:1.

#3.3 Experiment Formulation:

2.5 g of 50% soy PS in 125 mL sterile water (liposome was filteredthrough 5 μm filter) was mixed with 1.5 g of BSA or casein and thencombined with 536 mg of Amphotericin B (actually 500 mg based on thepotency assay with the concentration of 0.932 mg/mg) in 14.3 mL 0.1NNaOH solution with 3.57 g of castor oil to form liposomes containing theAmphotericin B. To make a stable geode formulation, 250 mg vitamin E in2.5 mL ethyl alcohol was then added into the mixture of geode liposome.The pH of the final mixture was adjusted to neutral with 0.3 mL 1N HCl.8.88 mL of 0.5M calcium chloride was then added to the resultant mixtureto form geode cochleates. Geode cochleates were then concentrated usinglyophilization to provide geode cochleates with sterile water in anyconcentrations (based on the experiment requirements) with alipld:Amphotericin B weight ratio of about 5:1.

Example 4 Amikacin Crystal Cochleate Formulation for In Vitro StudyProcedure of Amikacin Formulation for In Vitro Study: #4.1 ExperimentFormulation:

2 mg amikacin in 1.0 mL distilled deionized (D.D) water was filteredthrough 0.22 μm filter and combined with 20 mg of 50% soy PS in 2.0 mLsterile water (liposome was filtered through 5, 0.8 and 0.45 μm filter)to form liposomes containing the amikacin. To the resultant mixture wasthen added 0.206 mL of 0.1M calcium chloride to form cochleates. Themixture was then adjusting the drug concentration of the amikacin at 0.5mg/mL with sterile water. To make nice crystalline cochleates ofamikacin with a lipid:drug ratio of about 10:1.

#4.2 Experiment Formulation:

2 mg amikacin in 1.0 mL D.D water was filtered through 0.22 μm filterand combined with 20 mg of 50% soy PS in 2.0 mL sterile water (liposomewas filtered through 5, 0.8 and 0.45 μm filter) to form liposomescontaining the amikacin. To the resultant mixture was then added 0.206mL of 0.1M calcium chloride to form cochleates. To reduce theaggregation size of the crystals cochleates, 15 mg sodium chloride in 52μl sterile water was then added to the mixture of the cochleates. Themixture was then adjusting the concentration of the amikacin at 0.5mg/mL with sterile water. Also, the final product containing the 0.066Msodium chloride. To make nice crystalline cochleates of amikacin with alipid:drug ratio of about 10:1.

#4.3 Experiment Formulation:

2 mg amikacin in 1.0 mL D.D water was filtered through 0.22 μm filterand combined with 20 mg of 50% soy PS in 2.0 mL sterile water (liposomewas filtered through 5, 0.8 and 0.45 μm filter) to form liposomescontaining the amikacin. To the resultant mixture was then added 0.206mL of 0.1M calcium chloride to form cochleates. To reduce theaggregation size of the crystals cochleates, 76 mg sodium chloride in264 μl sterile water was then added to the mixture of the cochleates.The mixture was then adjusting the concentration of the amikacin at 0.5mg/mL with sterile water. Also, the final product containing 0.33Msodium chloride. To make nice crystalline cochleates of amikacin with alipid:drug ratio of about 10:1.

#4.4 Experiment Formulation:

2 mg amikacin in 1.0 mL D.D water was filtered through 0.22 μm filterand combined with 20 mg of 50% soy PS in 2.0 mL sterile water (liposomewas filtered through 5, 0.8 and 0.45 μm filter) to form liposomescontaining the amikacin. To the resultant mixture was then added 0.206mL of 0.1M calcium chloride to form cochleates. To reduce theaggregation size of the crystals cochleates, 152 mg sodium chloride in524 μl sterile water was then added to the mixture of the cochleates.The mixture was then adjusting the concentration of the amikacin at 0.5mg/mL with sterile water. Also, the final product containing 0.66Msodium chloride. To make nice crystalline cochleates of amikacin with alipid:drug ratio of about 10:1.

Example 5 Amikacin Crystal Cochleate Formulation for In Vivo StudyProcedure of Amikacin Formulation for In Vivo Study: #5.1 ExperimentFormulation:

200 mg amikacin in 20 mL D.D water was filtered through 0.22 μm filterand combined with 2000 mg of 50% soy PS in 200 mL sterile water(liposome was filtered through 5, 0.8 and 0.45 μm filter) to formliposomes containing the amikacin. To the resultant mixture was thenadded 17 mL of 0.1M calcium chloride to form cochleates. Cochleates werethen concentrated under lyophilization to provide crystal cochleates(about 6.7 mg/mL) with a lipid:amikacin ratio of about 10:1.

#5.2 Experiment Formulation:

200 mg amikacin in 20 mL D.D water was filtered through 0.22 μm filterand combined with 2000 mg of 50% soy PS in 200 mL sterile water(liposome was filtered through 5, 0.8 and 0.45 μm filter) to formliposomes containing the amikacin. To the resultant mixture was thenadded 17 mL of 0.1M calcium chloride to form cochleates. To reduce theaggregation size of crystals cochleates, 1125.2 mg sodium chloride in3.88 mL sterile water was then added to the mixture of the cochleates.Cochleates were then concentrated under lyophilization to providecrystal cochleates (about 6.7 mg/mL and 0.66M sodium chloride) with alipid:amikacin ratio of about 10:1.

Example 6 In Vitro Study of Aminoglycosides Crystal CochleateFormulation Macrophages and Infection

Cell lines: mouse peritoneal macrophage cell line (Raw 246.7), and/orTHP-1, a human macrophage cell line. Cells were cultured in DMEM andRPMI-1640, respectively, supplemented with 5% heat-inactivated fetalbovine serum. Macrophage monolayers were established by adding 10⁵macrophages to a 24-well tissue culture plate. After 24 hours,monolayers were infected and the infection was allowed to happen for 1hour, and then the extracellular bacteria were removed by washing. Someof the well contents were lysed and plated onto Middlebrook 7H10 agarplate, to determine the intracellular inoculum of the bacterium. Theremaining wells were treated daily with different aminoglycosides (i.e.,amikacin, gentamicin, and paromomycin) cochleates prepared in accordancewith the claimed method. After treatment, cell monolayers were lysed andthe lysate plated into 7H10 agar to quantify the intracellular load.

TABLE 1 Efficacy Comparison between Encochleated AminoglycosidesFormulation and Free Aminoglycosides Results Enhanced Efficacy vs. FreeDrug Amikacin Mycobacterium avium (10x-20x) Mycobacterium tuberculosis(7x) Francisella tularensis LVS (3x) Gentamicin Mycobacterium avium(10x) Mycobacterium smegmatis (50x) Mycobacterium tuberculosis (2x)Francisella tularensis LVS (2x) Francisella tularensis type A (4x)Paromomycin Francisella tularensis type A (4x) Cutaneous Leishmaniasis(3x)

As indicated in Table 1, aminoglycoside cochleates have enhancedefficacy against different bacteria compared to correspondingnon-encochleated free drugs.

Example 7 In Vitro Study of Amikacin Crystal Cochleate Formulation

Methods: As shown in Example 4, amikacin cochleate (Amkcch) formulationswere optimized for amikacin encochleation efficiency and particle sizeby varying the type of PS used, the PS:biological active ratio, PS:Ca⁺⁺ratio, and NaCl concentration. The efficacy of Amkcch againstintracellular Ma infections was evaluated in vitro using mouseperitoneal macrophage infected with M. avium strains MAC 101 or MAC 109.Mouse peritoneal macrophages (Mo) Raw 264.7 cells were seeded at 105cells/well. Mo monolayers were infected at ratio 1:10 for 1 h andextracellular bacteria removed. Monolayers were treated with freeamikacin and/or cochleate preparations for 4 days and the number ofintracellular bacteria determined Assays were repeated three times.

Results: Untreated control Ma strains grew within Mo from 3.8′ 105 to4.9′ 106. Ma within Mo treated with free amikacin (10 and 20 mg/mL) werekilled to 6.1 and 3.4′ 104 bacteria, respectively. Optimized Amkcch (10and 20 mg/mL) demonstrated greater than 10-fold enhanced efficacy,reducing bacterial count to 3.9 and 1.7′ 103 bacteria within Mo (p<0.05compared with free amikacin).

Conclusions: As indicated in the FIG. 1, amikacin cochleate formulationsare 10-50 fold more active than free amikacin against M. avium infectionin macrophage. Cochleate preparations of amikacin showed significant andenhanced activity against Ma strains in macrophages, suggesting thatcochleates achieved higher intracellular concentration for a longer timethan free amikacin.

Example 8 In Vivo Study of Amikacin Crystal Cochleate Formulation

A formation of amikacin cochleates has been developed. The in vivoefficacy amikacin cochleats against Mycobacterium avium complex (MAC)was evaluated using C57BL/6 black mice.

-   -   Mice, 12/group, were infected with M. avium 101 (8.1×107        bacterial/mouse) by tail vein injection.    -   After 7 days, 6 mice were harvested an the number of MAC in        spleen was quantified to establish the baseline bacterial load        (Time 0).    -   Mice were treated with various amikacin cochleates as indicated        in FIGS. 2 and 3    -   at 1.0 mg amikacin/day for 2 weeks.    -   Mice were harvested at week 3 and 2 days later (after 2 weeks of        treatment), and spleens homogenized and plated onto 7H10 agar.    -   Colonies on plates were counted and the data were analyzed.

As demonstrated in FIGS. 2 and 3, amikacin cochleates, given I.P. ororally, were active, reducing the number of bacterial load in thespleen. The amikacin cochleate preparation with high salt concentrationdoes orally was Conclusion: Oral delivery of Amikacin cochleateformulations demonstrates in vivo efficacy similar to IP free amikacin.

Example 9 High Salt Procedure for Aminoglycoside Formulations for InVitro Study #9.1 Experiment Formulation:

2 mg aminoglycoside in 1.0 ml D.D water was filtered through 0.22 μmfilter and combined with 20 mg of 50% soy PS in 2.0 ml sterile water(liposome was filtered through 5, 0.8 and 0.45 μm filter) to formliposomes containing the aminoglycoside. To the resultant mixture wasthen added 0.206 ml of 0.1M calcium chloride to form cochleates. Themixture was then adjusting the drug concentration of the aminoglycosideat 0.5 mg/ml with sterile water. To make nice crystalline cochleates ofaminoglycoside with a lipid:drug ratio of about 10:1.

#9.2 Experiment Formulation:

2 mg aminoglycoside in 1.0 ml D.D water was filtered through 0.22 μmfilter and combined with 20 mg of 50% soy PS in 2.0 ml sterile water(liposome was filtered through 5, 0.8 and 0.45 μm filter) to formliposomes containing the aminoglycoside. To the resultant mixture wasthen added 0.206 ml of 0.1M calcium chloride to form cochleates. Toreduce the aggregation size of the crystals cochleates, 15 mg sodiumchloride in 52 μl sterile water was then added to the mixture of thecochleates. The mixture was then adjusting the concentration of theaminoglycoside at 0.5 mg/ml with sterile water. Also, the final productcontaining the 0.066M sodium chloride. To make nice crystallinecochleates of aminoglycoside with a lipid:drug ratio of about 10:1.

#9.3 Experiment Formulation:

2 mg aminoglycoside in 1.0 ml D.D water was filtered through 0.22 μmfilter and combined with 20 mg of 50% soy PS in 2.0 ml sterile water(liposome was filtered through 5, 0.8 and 0.45 μm filter) to formliposomes containing the aminoglycoside. To the resultant mixture wasthen added 0.206 ml of 0.1M calcium chloride to form cochleates. Toreduce the aggregation size of the crystals cochleates, 76 mg sodiumchloride in 264 μl sterile water was then added to the mixture of thecochleates. The mixture was then adjusting the concentration of theaminoglycoside at 0.5 mg/ml with sterile water. Also, the final productcontaining 0.33M sodium chloride. To make nice crystalline cochleates ofaminoglycoside with a lipid:drug ratio of about 10:1.

#9.4 Experiment Formulation:

2 mg aminoglycoside in 1.0 ml D.D water was filtered through 0.22 μmfilter and combined with 20 mg of 50% soy PS in 2.0 ml sterile water(liposome was filtered through 5, 0.8 and 0.45 μm filter) to formliposomes containing the aminoglycoside. To the resultant mixture wasthen added 0.206 ml of 0.1M calcium chloride to form cochleates. Toreduce the aggregation size of the crystals cochleates, 152 mg sodiumchloride in 524 μl sterile water was then added to the mixture of thecochleates. The mixture was then adjusting the concentration of theaminoglycoside at 0.5 mg/ml with sterile water. Also, the final productcontaining 0.66M sodium chloride. To make nice crystalline cochleates ofaminoglycoside with a lipid:drug ratio of about 10:1.

TABLE 2 Results of Amikacin Encochleation for in House Study Obtainedfrom Amikacin- cochleation Supernatant (Degussa 85% vs Lipoid 50% Soy PSand before and after lyophilization) Deguss Deguss Lipoid 50% Lipoid 50%85% bSoy PS 85% bSoy PS Soy PS Soy PS Measured by Ratio of before Afterbefore After using the Lipid:Drug lyophilization lyophilizationlyophilization lyophilization Buffer 20:1   34% in sup 39.5% in sup34.5% in sup 33.8% in sup Buffer 10:1 69.1% in sup 72.6% in sup   50% insup 48.3% in sup Buffer  5:1 74.7% in sup 83.5% in sup 61.3% in sup58.1% in sup Degussa 85% 20:1 25.3% in sup 30.2% in sup 25.7% in sup  25% in sup soy PS Plain Sup Degussa 85% 10:1 55.4% in sup 58.4% in sup38.4% in sup 36.8% in sup soy PS Plain Sup Degussa 85%  5:1 61.6% in sup69.5% in sup 49.5% in sup   47% in sup soy PS Plain Sup Lipoid 50% 20:129.6% in sup 34.3% in sup   30% in sup 29.5% in sup Soy PS Plain SupLipoid 50% 10:1 59.8% in sup 62.9% in sup 43.6% in sup 42.4% in sup SoyPS Plain Sup Lipoid 50%  5:1 64.3% in sup 72.2% in sup 52.8% in sup50.8% in sup Soy PS Plain Sup

Encochleation percentage is determined by measuring the amount ofamikacin in the supernatant (ninhydrin assay) after forming thecochleates and then centrifuging. The amount in the supernatant is thensubtracted from the total amount during the encochleation process. Thisis reported as percent in supt. The lower the percent in supernatant thehigher the encochleation efficiency. The results listed in Table 2clearly demonstrate that 50% soy PS is more efficient at encochleatingthan 85% PS.

TABLE 3 Results of Amikacin Formulation Obtained from Absorbance at 400nm Ratio of Ratio of Ratio of Ratio of Ratio of 50% soy Ratio of 50% soyPS 85% soy 50% soy 85% soy PS to 85% soy Measured by to drug PS to drugPS to drug PS to drug drug PS to drug Using (10:1) (10:1) (20:1) (20:1)(5:1) (5:1) 50 mM 50.6% in 94.4% in 34.8% in 54.9% in 53.5% in 76.4% inPhosphate sup sup sup sup sup sup buffer + Amikacin 50% soyPS 41% in sup79% in sup 27.9% in 45.2% in 44.3% in 63.8% in Plain sup + sup sup supsup Amikacin 85% soyPS 44% in sup 82.8% in 30% in sup 47.8% in 46.8% in66.9% in Plain sup + sup sup sup sup Amikacin

Encochleation percentage is determined by measuring the amount ofamikacin in the supernatant (ninhydrin assay) after forming thecochleates and then centrifuging. The amount in the supernatant is thensubtracted from the total amount during the encochleation process. Thisis reported as percent in supt. The lower the percent in supernatant thehigher the encochleation efficiency. The results listed in Table 3clearly demonstrate that 50% soy PS is more efficient at encochleatingthan 85% PS.

TABLE 4 Results of Amikacin Formulation Obtained from Absorbance at 400nm Ratio of Measured by DOPS to Lipoid 50% Avanti 99% Using Amikacin SoyPS Avanti DOPS Soy PS NOF DOPS 50 mM 10:1 47.3% in sup 87.7% in sup84.3% in sup 86.4% in sup Phosphate buffer Lipoid 50% 10:1 37.1% in sup  77% in sup 73.5% in sup   64% in sup soy PS Plain cochleates supAvanti DOPS 10:1 43.6% in sup   83% in sup   80% in sup 81.7% in supplain sup Avanti 99% 10:1 41.9% in sup 84.6% in sup   81% in sup   83%in sup soy PS plain sup

Encochleation percentage is determined by measuring the amount ofamikacin in the supernatant (ninhydrin assay) after forming thecochleates and then centrifuging. The amount in the supernatant is thensubtracted from the total amount during the encochleation process. Thisis reported as percent in supt. The lower the percent in supernatant thehigher the encochleation efficiency. The results listed in Table 4clearly demonstrate that 50% soy PS is more efficient at encochleatingthan 99.99% PS.

FIG. 4 shows Gentamicin Encochleation Related to PS Properties. Similarto Tables 1-3, the results listed in FIG. 4 clearly demonstrate that 50%soy PS is more efficient at encochleating gentamicin at lower bile saltconcentrations than 85% PS or 99.99% PS.

Example 10 Encochleation Efficiency of Amikacin, Gentamicin, andParomomycin Bile Salt Cochleates

#10.1 Procedure for Aminoglycoside Formulation with Bile Salts (afterCalcium) for In Vitro Studies.

Crystalline cochleates of aminoglycoside formulation with differentamount of bile salts (Sigma Aldrich catalog #48305 Fluka cholic acidsodium salt, ˜50% deoxycholic acid sodium salt, ˜50%):

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. To theresultant mixture, 0.159 ml of 0.1M calcium chloride was then added withvigorous mixing to form aminoglycoside cochleates. In order to makeparticle size of the aminoglycoside cochleate crystals smaller, 20.4 mgbile salts were then added to the mixture of the aminoglycosidecochleates. The mixture was then adjusted to a drug concentration of theaminoglycoside at 0.5 mg/ml with the buffer as the suspension. The finalaminoglycoside cochleate formulations contained 11.3 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. To theresultant mixture, 0.159 ml of 0.1M calcium chloride was then added withvigorous mixing to form aminoglycoside cochleates. In order to makeparticle size of the aminoglycoside cochleate crystals smaller, 13.6 mgbile salts were then added to the mixture of the aminoglycosidecochleates. The mixture was then adjusted to a drug concentration of theaminoglycoside at 0.5 mg/ml with the buffer as the suspension. The finalaminoglycoside cochleate formulations contained 7.8 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. To theresultant mixture, 0.159 ml of 0.1M calcium chloride was then added withvigorous mixing to form aminoglycoside cochleates. In order to makeparticle size of the aminoglycoside cochleate crystals smaller, 6.8 mgbile salts were then added to the mixture of the aminoglycosidecochleates. The mixture was then adjusted to a drug concentration of theaminoglycoside at 0.5 mg/ml with the buffer as the suspension. The finalaminoglycoside cochleate formulations contained 3.9 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. To theresultant mixture, 0.159 ml of 0.1M calcium chloride was then added withvigorous mixing to form aminoglycoside cochleates. In order to makeparticle size of the aminoglycoside cochleate crystals smaller, 3.4 mgbile salts were then added to the mixture of the aminoglycosidecochleates. The mixture was then adjusted to a drug concentration of theaminoglycoside at 0.5 mg/ml with the buffer as the suspension. The finalaminoglycoside cochleate formulations contained 1.95 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. To theresultant mixture, 0.159 ml of 0.1M calcium chloride was then added withvigorous mixing to form aminoglycoside cochleates. In order to makeparticle size of the aminoglycoside cochleate crystals smaller, 1.7 mgbile salts were then added to the mixture of the aminoglycosidecochleates. The mixture was then adjusted to a drug concentration of theaminoglycoside at 0.5 mg/ml with the buffer as the suspension. The finalaminoglycoside cochleate formulations contained 0.97 mM.

#10.2 Procedure of Aminoglycoside Formulation with Bile Salts (BeforeCalcium) for In Vitro Studies.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. Inorder to make particle size of the aminoglycoside cochleate crystalssmaller, 20.4 mg bile salts were then added to the mixture of theaminoglycoside liposomes. To the resultant mixture, 0.159 ml of 0.1Mcalcium chloride was then added with vigorous mixing to formaminoglycoside cochleates. The mixture was then adjusted to a drugconcentration of the aminoglycoside at 0.5 mg/ml with same the buffer asthe suspension. The final aminoglycoside cochleate formulationscontained 11.3 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. Inorder to make particle size of the aminoglycoside cochleate crystalssmaller, 13.6 mg bile salts were then added to the mixture of theaminoglycoside liposomes. To the resultant mixture, 0.159 ml of 0.1Mcalcium chloride was then added with vigorous mixing to formaminoglycoside cochleates. The mixture was then adjusted to a drugconcentration of the aminoglycoside at 0.5 mg/ml with the buffer as thesuspension. The final aminoglycoside cochleate formulations contained7.8 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. Inorder to make particle size of the aminoglycoside cochleate crystalssmaller, 6.8 mg bile salts were then added to the mixture of theaminoglycoside liposomes. To the resultant mixture, 0.159 ml of 0.1Mcalcium chloride was then added with vigorous mixing to formaminoglycoside cochleates. The mixture was then adjusted to a drugconcentration of the aminoglycoside at 0.5 mg/ml with the buffer as thesuspension. The final aminoglycoside cochleate formulations contained3.9 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. Inorder to make particle size of the aminoglycoside cochleate crystalssmaller, 3.4 mg bile salts were then added to the mixture of theaminoglycoside liposomes. To the resultant mixture, 0.159 ml of 0.1Mcalcium chloride was then added with vigorous mixing to formaminoglycoside cochleates. The mixture was then adjusted to a drugconcentration of the aminoglycoside at 0.5 mg/ml with the buffer as thesuspension. The final aminoglycoside cochleate formulations contained1.95 mM.

Aminoglycoside, 2 mg in 0.2 ml sterile water was filtered through a 0.22μm filter and combined with 20 mg of 50% soy PS liposome in 2.0 mlsterile water (the soy PS liposome was first filtered through 5, 0.8 and0.45 μm filters) to form liposomes containing the aminoglycoside. Inorder to make particle size of the aminoglycoside cochleate crystalssmaller, 1.7 mg bile salts were then added to the mixture of theaminoglycoside liposomes. To the resultant mixture, 0.159 ml of 0.1Mcalcium chloride was then added with vigorous mixing to formaminoglycoside cochleates. The mixture was then adjusted to a drugconcentration of the aminoglycoside at 0.5 mg/ml with the buffer as thesuspension. The final aminoglycoside cochleate formulations contained0.97 mM.

FIGS. 5-11 show encochleation efficiency of aminoglycoside bile salts,including amikacin bile salts, gentamycin bile salts, and paromomycinbile salts.

Encochleation percentage is determined by measuring the amount ofaminoglycoside bile salts in the supernatant (ninhydrin assay) afterforming the cochleates and then centrifuging. The amount in thesupernatant is then subtracted from the total amount during theencochleation process. These results clearly demonstrate that 50% soy PSis more efficient at encochleating at lower bile salt concentrationsthan 75% PS or 99.99% PS. A is bile salts added after the encochleation.B is bile salts added before the encochleation.

1-35. (canceled)
 36. A crystal cochleate, comprising: a soy-basedphospholipid comprising 50% by weight soy phosphatidylserine, calcium,and amphotericin B.
 37. A geode cochleate, comprising: a soy-basedphospholipid that comprises 50% by weight soy phosphatidylserine,calcium, an oil, and amphotericin B
 38. The crystal cochleate of claim6, wherein the ratio of the soy-based phospholipid to amphotericin B isabout 5:1.
 39. The geode cochleate of claim 37, wherein the ratio of thesoy-based phospholipid to amphotericin B is about 5:1.
 40. A method ofmaking the crystal cochleate of claim 36, wherein the method comprises:(a) dissolving amphotericin B in a sodium hydroxide solution to form abasic amphotericin B solution; (b) mixing the basic amphotericin Bsolution and the soy-based phospholipid comprising 50% by weight soyphosphatidylserine to form liposomes in an aqueous solution, wherein theliposomes contain amphotericin B; and (c) adding calcium to theliposomes to form the crystal cochleate.
 41. The method of claim 40,wherein the ratio of the soy-based phospholipid to amphotericin B isabout 5:1.
 42. The method of claim 40, wherein the aqueous solution is aphosphate buffered solution.
 43. The method of claim 40, furthercomprising adding an antioxidant to the liposomes before adding thecalcium.
 44. The method of claim 43, further comprising lyophilizing thecrystal cochleate.
 45. A method of making the geode cochleate of claim37, wherein the method comprises: (a) dissolving amphotericin B in asodium hydroxide solution to form a basic amphotericin B solution; (b)mixing the basic amphotericin B solution with the oil and the soy-basedphospholipid comprising 50% by weight soy phosphatidylserine to formgeode liposomes in an aqueous solution, wherein the geode liposomescontain amphotericin B; and (c) adding calcium to the geode liposomes toform the geode cochleate.
 46. The method of claim 45, wherein the ratioof the soy-based phospholipid to amphotericin B is about 5:1.
 47. Themethod of claim 45, further comprising adding an antioxidant to theliposomes containing the amphotericin B before adding the calcium. 48.The method of claim 45, farther comprising adjusting the pH of the geodeliposomes of step (b) or the geode cochleate of step (c) to neutral. 49.The method of claim 45, further comprising lyophilizing the geodecochleate.